Ecteinascidins 736 and 722

ABSTRACT

Ecteinascidins 722 and 736 (Et&#39;s 722 and 736) have been isolated from the Caribbean tunicate Ecteinascidia turbinata and their structures have been assigned as tetrahydro- beta -carboline-substituted bis(tetrahydro- isoquinolines) related to the previously reported Et&#39;s 729 and 743. Et&#39;s 722 and 736 protect mice in vivo at very low concentrations against P388 lymphoma, B16 melanoma, and Lewis lung carcinoma.

STATEMENT OF GOVERNMENT SUPPORT

This invention was supported in part by a grant from the NationalInstitute of Allergy and Infectious Diseases (No. AI04769). Mass spectrawere obtained in the Mass Spectrometry Laboratory, School of ChemicalSciences, University of Illinois, and supported in part by a grant fromthe National Institute of General Medical Sciences (No. GM27029).

CROSS REFERENCE TO RELATED APPLICATION

This application describes compounds related to those described incopending U.S. Pat. Application Ser. No. 07/548.060. filed Jul. 5, 1990,the disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Rinehart et al. have recently reported on ecteinascidins (Et's) 729(hydrated molecular weight, 747), 743 (761), 745. 759A,B (777). 770, andtheir derivatives O-methyl-Et 729 and O-methyl-Et 743. See for example,J. Orz. Chem., 1990, 55, 4512-4515; Topics in Pharmaceutical Sciences1989, Amsterdam Medical Press, 1989, pp. 613-626; J. Nat. Prod., 1990,53, 771-792; Biological Mass. Spectrometry, Elsevier 1990, pp. 233-258;and Pure Appl. Chem., 1990, 62, 1277-1280. Two of those compounds (Et729, 743) have also been described by others. (See for example, Wrightet al., J. Org. Chem., 1990, 55, 4508-4512).

The major component, ecteinascidin 743 (Et 743, (Rinehart et al.. J.Orz. Chem., 1990, 55, 4512-4515), and the others were assignedtris(tetrahydroisoquinoline) structures by correlation NMR techniques,as well as by fast atom bombardment (FAB)MS and tandem MS (FABMS/MS).Among these potent antitumor agents, Et 729 showed especially promisingactivities vs. tumor cells, but only minute quantities of pure samplewere obtained. See for example, Rinehart et al., Topics inPharmaceutical Sciences 1989 pp. 613-626, Amsterdam Medical Press B.V.,The Netherlands, (1989), Holt et al., Diss. Abstr. Int. B. 47, 3771-3772(1987) and Rinehart et al., U.S. Pat. Appln. Ser. No. 872,189, filedJun. 9, 1986; PCT Intl. Appln. W087 07,610, filed Dec. 17, 1987; Chem.Abstr., 109, 811j, (1988).

The need for further biological evaluation promoted the development of amore efficient large-scale isolation procedure. During that process, twonew biologically active ecteinascidins; Et 736 (754) and Et 722 (740),were isolated from Ecteinascidia turbinata samples collected at variouslocations in the Caribbean.

SUMMARY OF THE INVENTION

The present invention is directed to the isolation of two new compounds,Et 736 and 722 from E. turbinata, together with assignment of theirstructures and biological activities. The data reported herein supportour previously proposed biogiogenetic pathway. See, Rinehart et al., J.Org. Chem., supra.

Thus, the present invention is directed to the following new compounds3, 4 and 5. Compound 1 and 2, Et 743 and 729, respectively, are shownfor comparison purposes. ##STR1##

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A (top) is the mass spectrum MS/MS (FAB), on a fragment ion at m/z493 of Et 743.

FIG. 1B (bottom) is the mass spectrum MS/MS (FAB), on a fragment ion atm/z 493 of Et 736.

FIGS. 2 and 3 are graphs illustrating the percentage of inhibition ofthe growth of L1210 cells by Et 736 (- - -) and Et 722 ( ). Also shownon the graphs for comparison are the inhibitory effects of Et 743 ( . ..) and Et 729 ( ).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Samples of E. turbinata were collected in the Florida Keys and Belize,and their cytotoxic extracts were separated by using solvent partition,countercurrent chromatography (CCC), (see for example, Y. Ito, CRC Crit.Rev. Anal. Chem., 17, 65-143, (1986)) and normal and reversed-phase (RP)gravity columns. Final purifications were carried out by (C-18) RP-HPLC.

All samples examined contained Et's 743 (761), 729 (747), 736 (754), and722 (740) in various proportions. Et 736 (754), [α]_(D) = 76°(c 0.53,CHCl₃), showed "molecular" ions at m/z 753.2588 (C₄₀ H₄₁ N₄ O₉ S, Δ0.6mmu, M - H, negative ion HRFABMS). Et 722 (740), [α]_(D) =-40° (c 1.64,CHCl₃), showed "molecular" ions at m/z 739.2433 C₃₉ H₃₉ N₄ O₈ S, Δ-0.7mmu, M +H -H₂ O, positive ion HRFABMS). ¹ H and ¹³ C NMR spectra for Et722 (740) vis-a-vis Et 736 (754) lacked an N¹² --CH₃ signal and showedan upfield shift for the adjacent carbons C-11 and C-13 (Table 1),indicating that Et 722 (740) was the N¹² -demethyl derivative of Et 736(754).

Comparison of NMR data (Table 1) for these new compounds with those forEt 743 (761) and Et 729 (747) indicated that thebis(tetrahydroisoquinoline) units A and B are the same in Et 736 (754)and Et 722 (740) as in the earlier Et's. This was also supported by NMRcorrelation spectroscopy data, including COSY, phase-sensitive COSY,CSCM, and COLOC sequences, although some of the expected correlationswere missing due to the broad peaks observed.

                                      TABLE 1                                     __________________________________________________________________________    .sup.1 H and .sup.13 C NME Data for Et's 743, 729, 736, and 722 (1-4) in      CD.sub.3 OD--CDCl.sub.3 (3:1)                                                 Chemical shift (δ), multiplicity.sup.a (j in Hz)                        Carbon                                                                        or   1                2           3              4                            Proton.sup.b                                                                       .sup.13 C                                                                          .sup.1 H    .sup.13 C                                                                          .sup.1 H                                                                             .sup.13 C.sup.c                                                                    .sup.1 H  .sup.13 C                                                                          .sup.1 H                __________________________________________________________________________    1     56.3, d                                                                           4.78, br s   56.8, d                                                                           4.69, br s                                                                            54.8, d                                                                           4.71, br s                                                                               56.7,                                                                             4.72, br s              3     58.8, d                                                                           3.72.sup.c   57.1, d                                                                           3.72 br d (5.5)                                                                       57.8, d                                                                           3.76, br s                                                                               58.5,                                                                             3.53, d (4.5)           4     42.7, d                                                                           4.58, br s   42.5, d                                                                           4.58, br s                                                                            42.3, d                                                                           4.58, br s                                                                               43.1,                                                                             4.50, br s              5    142.2, s         142.3, s    140.8, s       141.9, s.sup.d               6    113.9, s         114.0, s    112.6, s       113.4, s                     7    146.5, s.sup.d   146.6, s.sup.d                                                                            145.4, s       146.8, s                     8    141.9, s         141.5, s    140.5, s       142.1, s.sup.d               9    116.0, s         115.5, s    115.4, s       115.9, s                     10   122.0, s         121.4, s    120.9, s       121.8, s                     11    55.6, d                                                                           4.40, br d (3.5)                                                                           47.8, d                                                                           4.73 (5.0)                                                                            54.5, d                                                                           4.73, br s                                                                               48.0,                                                                             4.44, d (4.8)           13    54.0, d                                                                           3.12, br s   47.2, d                                                                           3.94, d (10.0)                                                                        52.6, d                                                                           3.90, br s                                                                               46.9,                                                                             3.57, br d (9.0)        14    24.5, t                                                                           2.51, 2H, br d (4.5)                                                                       25.1, t                                                                           3.22, d (18.0)                                                                        23.2, t                                                                           3.30.sup.c                                                                               27.9,                                                                             3.15, d (17.7)                                     3.12, dd (9.8,                                                                            3.08, dd (10.0, 19.0)                                                                        3.01, dd (17.7,                                                               9.3)                                               18.0)                                              15   120.9, d                                                                           6.15, s     121.2, d                                                                           6.62, s                                                                              120.3, d                                                                           6.70, s   121.0,                                                                             6.59, s                 16   131.2, s         130.6, s    130.3, s       131.4, s                     17   145.1, s         144.9, s    143.0, s       144.4, s                     18   149.8, s         148.6, s    148.2, s       148.2, s                     19   119.2, s         120.2, s    118.5, s       124.3, s                     20   131.5, s         132.7, s    130.9, s       132.2, s                     21    92.1, d                                                                           4.26, d (3.0)                                                                              90.1, d                                                                           4.33, d (3.0)                                                                         91.5, d                                                                           4.46, d (2.4)                                                                            91.1,                                                                             4.12, s                 22    61.2, t                                                                           5.14, d (11.0)                                                                             61.5, t                                                                           5.15, d (11.0)                                                                        62.0, t                                                                           5.20, dd (12.5, 0.5)                                                                     61.6,                                                                             5.17, d (11.1)                    4.09, dd (11.0, 2.0)                                                                           4.11, dd (2.5,                                                                            4.18, dd (12.5, 1.5)                                                                         4.14, dd (11.4,                                                               1.2)                                               11.0)                                              OCH.sub.2 O                                                                        103.1, t                                                                           6.07, d (1.0)                                                                             103.1, t                                                                           6.09, d (0.5)                                                                        101.7, t                                                                           6.26, d (1.0)                                                                           103.1,                                                                             6.21, d (1.0)                     5.98, d (1.0)    6.00, d (0.5)                                                                             6.07, d (1.0)  6.04, d (1.0)            1'   65.3, s          65.2, s     61.9, s        63.1, s                      3'   40.3, t                                                                           3.13, dt (11.0, 4.0)                                                                       40.4, t                                                                           3.12, m                                                                               39.6, t                                                                           3.30.sup.c                                                                               40.6,                                                                             3.30, m                           2.77, ddd (3.5, 5.5, 11.0)                                                                     2.77, m     2.90, dt (11.5, 4.5)                                                                         2.86, m                  4'   28.6, t                                                                           2.60, ddd (5.5, 10.5, 16.0)                                                                28.6, t                                                                           2.60, ddd (5.5,                                                                       20.9, t                                                                           2.63, m (2H)                                                                             21.6,                                                                             2.61, m (2H)                      2.42, ddd (3.5, 3.5, 16.0)                                                                     10.5, 16.0)                                                                   2.42, ddd (3.5,                                                               3.5, 16.0)                                          5'  115.6, d                                                                           6.38, s     115.7, d                                                                           6.39, s                                                                              116.9, d                                                                           7.33, d (8.0                                                                            118.8, d                                                                           7.31, d (7.8)            6'  146.4, s.sup.d   146.4, s.sup.d                                                                            117.7, d                                                                           6.92, dt (8.0)                                                                          119.6,                                                                             6.91, dt (0.9,                                                                7.8)                     7'  146.4, s.sup.d   146.4, s.sup.d                                                                            120.7, d                                                                           7.12, dt (0.6, 8.0)                                                                     122.5,                                                                             7.00, dt (0.9,                                                                7.8)                     8'  111.3, d                                                                           6.42, br s  111.3, d                                                                           6.41, br s                                                                           111.7, d                                                                           7.29, d (0.6, 8.0)                                                                      111.9,                                                                             7.21, d (7.8)            9'  125.4, s         125.2, s    126.6, s       127.3, s                     10'  128.8, s         129.0, s    135.6, s       137.4, s                     11'  173.1, s         173.2, s    171.2, s       172.5, s                     12'   43.1, t                                                                           2.33, br d (15.5)                                                                          42.7, t                                                                           2.40     38.9, t                                                                          2.78, d (15.6)                                                                           39.9,                                                                             2.74, d (15.0)                    2.05.sup.f       2.07.sup.f  2.15, br d (15.3)                                                                            2.12, br d (15.0)       13'                               129.3, s       130.8, s                     14'                               109.5, s       109.8, s                     5 C = O                                                                            169.8, s         169.8, s    169.5, s       170.7, s                     5 OAc                                                                               20.5, q                                                                           2.29, s      20.5, q                                                                           2.30, s                                                                               20.3, q                                                                           2.28, s    20.6,                                                                             2.27, s                 6 CH.sub.3                                                                          9.9, q                                                                            2.01, s      9.8, q                                                                            2.02, s                                                                               9.4, q                                                                            2.02, s    9.7, q                                                                            2.01, s                 16 CH.sub.3                                                                         16.1, q                                                                           2.28, s      16.1, q                                                                           2.29, s                                                                               15.7, q                                                                           2.37, s    16.2,                                                                             2.32, s                 17 OCH.sub.3                                                                        60.2, q                                                                           3.73, s      60.3, q                                                                           3.71, s                                                                               60.0, q                                                                           3.76, s    60.3,                                                                             3.72, s                 7' OCH.sub.3                                                                        55.7, q                                                                           3.58, s      55.6, q                                                                           3.58, s                                            12 NCH.sub.3                                                                        41.1, q                                                                           2.23, s                  40.9, q                                                                           2.49, br s                             __________________________________________________________________________     .sup.a s = singlet, d = doublet, t = triplet, q = quartet, br = broad.        .sup.b Proton assignments are based on COSY and homonuclear decoupling        experiments; carbon multiplicities were determined by APT and DEPT            spectra. Carbons for 4 were assigned by analogy to those of 3. .sup.c         CD.sub.3 OD--CDCl.sub.3, 7:1, .sup.d Assignments are interchangeable.         .sup.e Signals overlap the solvent peak. .sup.f Signals overlap the methy     singlet.                                                                 

Further support for A-B units' identity was provided by HRFABMS (TableII) and FABMS/MS (Scheme I) fragmentation patterns. Importantfragmentation ions for the A-B bis(tetrayhydroisoquinoline) unitobserved for Et 743 were also seen for Et 736 (Table II).

                  TABLE II                                                        ______________________________________                                        Comparison of HRFABMS Fragmentation Data for                                  Et 743 and Et 736                                                             Observed ions                                                                 Et 743  Et 736    Formula      Fragment                                       ______________________________________                                        744.2648          C.sub.39 H.sub.42 N.sub.3 O.sub.10 S                                                       (M + H - H.sub.2 O)                                    737.2655  C.sub.40 H.sub.40 N.sub.4 O.sub.8 S                                                        (M + H - H.sub.2 O)                            523.2011                                                                              523.1960  C.sub.28 H.sub.31 N.sub.2 O.sub.8                                                          a + 2H                                         495.2126                                                                              495.2126  C.sub.27 H.sub.31 N.sub.2 O.sub.7                                                          b + 2H                                         493.1980                                                                              493.1980  C.sub.27 H.sub.29 N.sub.2 O.sub.7                                                          b                                              477.1978                                                                              477.2024  C.sub.27 H.sub.29 N.sub.2 O.sub.6                                                          c                                              463.1837                                                                              463.1862  C.sub.26 H.sub.27 N.sub.2 O.sub.6                                                          d                                              218.1174                                                                              218.1180  C.sub.13 H.sub.16 NO.sub.2                                                                 g                                              204.1027                                                                              204.1025  C.sub.12 H.sub.14 NO.sub.2                                                                 h - H                                          ______________________________________                                         ##STR2##

In addition, the tandem FAB mass spectra of the key fragment ion m/z 493for Et 736 and Et 743 were essentially identical (see FIGS. 1A and 1B),arguing that these Et's contain the same bis(tetrahydro-isoquinoline)subunit.

Addition of 5 μL (ca. 10 equiv.) of methanol-d₄ to a CDCl₃ solution ofEt 736 gave a drastic downfield change in chemical shift for C-21 (δ81→90) due to the chemical exchange of OH at C-21 by OCD₃, just as in thecase of Et 743 (67 82 →92), see, Rinehart et al., J. Org. Chem.. 1990,55, 4512-4515. Similarly, treatment of Et 736 with methanol at roomtemperature and evaporation of the solvent gave O-methyl-Et 736 (754)(5, M - H at 767.2761 for C₄₁ H₄₃ N₄ O₉ S, Δ0.1 mmu, negative ionHRFABMS), which sh showed a new methoxyl signal (δ53.8, CDCl₃) in its hu13C NMR spectrum.

Subtraction of the bis(tetrahydroisoquinoline) unit (A-B) from themolecular formula for Et 736 (754) gives the formula C₁₃ H₁₂ N_(2O) ₂ Sfor the rest of the molecule (unit C). The ¹³ C NMR signals for thissubunit include one carbonyl carbon and eight aromatic/olefinic carbons,leaving three rings for the structure. The UV spectrum (MeOH) λ_(max)292 (ξ11 900), 283 (12 500), 221 (sh 44 800), 207 (11 900) nm along with¹³ C NMR resonances at δ109.5 and 129.3 suggest this structural unit tobe a tetrahydro- β-carboline; (see, Shamma et al., Carbon-13 NMR ShiftAssignments of Amines and Alkaloids: Plenum Press, New York. (1979) andNakagawa et al., J. Am. Chem. Soc., 111. 2721-2722 (1989) and Rinehartet al., J. Am. Chem. Soc., 23, 3290-3297, (1984)), this was confirmed byCOSY spectra showing the aromatic spin system of an ortho-disubstitutedbenzene ring with signals from δ7.32 to 6.91, as well as an aliphatic--CH₂ --CH₂ --X spin system (Scheme II). NMR data for unit C closelyresemble those of the dihydro-β-carboline debromoeudistomin L. (See,Nakagawa et al., J. Am. Chem. Soc., 111, 2721-2722 (1989). The remainingatoms in unit C --a carbonyl, a CH₂, and a sulfur atom - can beassembled as shown in Scheme II to be consistent with the chemicalshifts for C-11' and C-12' in Et 743 (see, Table I).

HRMS data on fragmentation ions at m/z 216 and 243, which were seen bothin FAB and tandem FAB mass spectra, also supported this assignment(Scheme I). A COLOC spectrum showing a long-range correlation betweenC-11' and a proton on C-22, along with an IR (CCl₄) absorption at 1753cm⁻¹, agreed with an ester linkage between C-11'(carbonyl) and C-22.

The molecular formula C₄₀ H₄₂ N₄ O₉ S for Et 736 (754) requires 22degrees of unsaturation, one more than assigned thus far. The additionalring required is consistent with the ¹³ C NMR chemical shifts only if itis formed between the sulfur and C-4 of the isoquinoline B ring, as seenin the Et 743 series. Consequently, the structures of Et 736 and 722were assigned as 3 and 4. These compounds are closely relatedbiogenetically to those of the Et 743 series, except for theirtetrahydro-β-carboline portion, which presumably comes from tryptamineinstead of dopamine, (see, Rinehart et al., J. Org. Chem.. 55,4512-4512, (1990)). Indeed, the water-soluble portion of the sametunicate extract yielded tryptamine itself, also supporting thisbiogenetic proposal.

The bioactivities of Et's 722 and 736 appear to be comparable to thoseof Et's 729 and 743. Et's 722 and 736 inhibit L1210 leukemia cells tothe extent of 90% in plate assays at 2.5 and 5.0 ng/mL, respectively(see, FIGS. 2 and 3). More importantly, Et 722 is highly active in vivo,giving T/C 230 (4/6 survivors) at 25 μg/kg day vs. P388 murine leukemia,T/C 200 at 50 μg/kg day vs. B16 melanoma, and T/C 0.27 at 50 μg/kg dayvs. Lewis lung carcinoma (see, Table III).

                                      TABLE III                                   __________________________________________________________________________    Activity in vivo of Ecteinascidins 729 and 722                                P388 lymphocyclic             Lewis lung carcinoma                            Dose  leukemia     B16 melanoma  Mean tumor                                   μg/Kg/mi                                                                         T/C.sup.a                                                                          Survivors (day)                                                                       T/C.sup.a                                                                        Survivors (day)                                                                       T/C.sup.b                                                                        volume (ram.sup.3)                           __________________________________________________________________________    Control                                                                             100  0(12)   100                                                                              0       1.00                                                                             1512                                         Et 729                                                                        25.0  130  0(13)    76                                                                              0(42)   0.00                                                                              2                                           12.5  190  2(21)   253                                                                              5/10(42)                                                                              0.04                                                                              57                                           6.25 NT   NT      197                                                                              0(37)   0.14                                                                             216                                          Et 722                                                                        50.0  150  1(23)   200                                                                              0(36)   0.27                                                                             412                                          25.0  >230 4(23)   185                                                                              0(35)   0.62                                                                             934                                          12.0  205  0(23)   156                                                                              0(30)   0.87                                                                             1319                                         __________________________________________________________________________     Significant activity: .sup.a T/C ≧ 125; .sup.b T/C ≦ 40.   

Additional data supporting the activity of Et 722 are shown in thefollowing tables:

    __________________________________________________________________________    TUMOR GROWTH INHIBITION - Day 14                                                       Tumor: 816            Species: Mouse                                          Generation: 77801     Strain: 8DF1                                            Tissue: SOLID TUMOR   Male                                                    Level: 1:10 BREI      Source: Charles River Kingston                          Site: 0.5 ml, Sc.     DOB:                                                             Mean Body Wt                                                                          Median       Mean                                         Dose  Schedule                                                                            Change (grams)                                                                        N.P.                                                                             Tumor Vol.                                                                              Tumor Vol.                                                                           ST                              Compound                                                                            ug/kg/inJ                                                                           & Route                                                                             Day 1-5 D-14                                                                             (mm 3) T/C                                                                              (mm 3) DEV T/C                                                                              T                        __________________________________________________________________________    ET 722                                                                              50.00 QD1-9, 1P                                                                           -0.5    0  395    0.29                                                                              412*  157 0.27                                                                             14,862                         25.00       1.0     0  908    0.66                                                                             934    158 0.82                                                                             7,588                          12.50       1.9     0  1204   0.88                                                                             1319   509 0.87                                                                             1,420                    __________________________________________________________________________     N.P. = # of Nonpalpable Tumors on Day 14                                      *Significant Activity: T/C< = 0.40 and p = <0.01 By t Test               

    __________________________________________________________________________    Interim Results: Day 23                                                       ANTI-TUMOR ACTIVITY VS. P388 LYMPHOCYTIC LEUKEMIA                                      Tumor: 816            Species: Mouse                                          Generation: 77801     Strain: 8DF1                                            Tissue: SOLID TUMOR   Male                                                    Level: 1:10 BREI      Source: Charles River Kingston                          Site: 0.5 ml, Sc.     DOB:                                                             Body Wt.        Mean     Median   Alive                           Dose  Schedule                                                                            Change (gm)     Survival Survival Day                       Compound                                                                            ug/kg/inJ                                                                           & Route                                                                             Day 5  Day of Death                                                                           Time % T/C                                                                             Time % T/C                                                                             23                        __________________________________________________________________________    ET 722                                                                              50.00 QD1-9, 1P                                                                           -0.3   10 13 15 15 21                                                                         14.8 145*                                                                              15.0 150 1                               25.00       0.2    13 21             >23  >230                                                                              4                               12.50       0.4    14 19 20 21 22 22                                                                      19.7 199*                                                                              20.5 205 0                         __________________________________________________________________________     N.P. = # of Nonpalpable Tumors on Day 14                                      *Significant Activity: T/C< = 0.40 and p = <0.01 By t Test               

    __________________________________________________________________________    Interim Results: Day 42                                                       ANTI-TUMOR ACTIVITY VS. 816 MELANOMA                                                      Tumor: 816            Species: Mouse                                          Generation:           Strain: 8DF1                                            Tissue: BREI          Sex: Male                                               Level: 1:10; 0.5 cc   Source: Charles River Kingston                    Dose  Schedule                                                                            Change (gm)           Survival  Survival Day                Compound                                                                            ug/kg/inJ                                                                           & Route                                                                             Day 5   Day of Death  Time % T/C                                                                              Time %                                                                                 24C                __________________________________________________________________________    ET 722                                                                              50.00 QD1-9,1P                                                                            -1.1    32 32 33 34 34 34 34 35 35 36                                                               33.9 185* 34.0 200 0                        25.00       0.9     28 30 30 30 31 32 34 34 35 35                                                               31.9 183* 31.5 185 0                        12 50       0.8     17 18 23 23 26 27 27 29 30 30                                                               25.0 144* 28.5 155 0                  __________________________________________________________________________     816 (0.5 ml, 1:10 brie) implanted ip into male BDF1 mice on day 0,            compounds dissolved or suspended in sterile 0.9% NaCl solution (plus          minimal amounts of ethanol and Tween80 as needed) and administered ip day     1-9 in a volume of 0.5 ml/mouse. Mice were weighed days 1 and 5 and death     were recorded daily.                                                          *Significant activity: T/C> = 125%                                       

It seems especially promising that some in vivo selectivity isdemonstrated by the ecteinascidins; Et 722 is more active than Et 729vs. P388 (T/C 190 at 12.5 μ/kg day for 729) but less active against B16(T/C 253 for 729).

The present invention will be further illustrated with reference to thefollowing examples which aid in the understanding of the presentinvention, but which are not to be construed as limitations thereof. Allpercentages reported herein, unless otherwise specified, are percent byweight. All temperatures are expressed in degrees Celsius.

GENERAL

IR spectra were recorded on an IBM IR/32 FTIR spectrophotometer. Opticalrotations were measured with a DIP 370 digital polarimeter with a sodiumlamp (589 nm) and 5 cm (1 mL) cell. Melting points were measured with amelting point apparatus and were not corrected. NMR spectra wereobtained with QE 300 and GN 500 spectrometers. High- and low-resolutionFAB mass spectra and FABMS/MS data were measured on a 70-SE-4Fspectrometer. Gravity columns were prepared with silica gel (70-230mesh) or RP C-18 silica gel (Martex 20-40 μ or Fuji-Division 100 -200μ). An Ito multi-layer coil separator-extractor was used for CCC, (Y.Ito, CRC Crit. Rev. Anal. Chem., 17, 65-143, (1986).

EXAMPLE 1

Collection and Extraction. -- A sample (19 kg), collected in the FloridaKeys in Aug., 1989, and immediately frozen on site, was stored at -20°C. until use. The defrosted sample was squeezed gently by hand, and thesolid material was soaked in 2-propanol (4 L ×3). The alcoholic extractwas separated by decantation from the solid and concentrated to anaqueous emulsion, which was then extracted with CH₂ Cl₂ (0.5 L ×8). TheCH₂ Cl₂ extract was concentrated to a crude oil (20.2 g).

EXAMPLE 2

Separation and Purification. -- All separations were monitored bybioassays against L1210 murine leukemia cells and Micrococcus luteus.The crude extract was partitioned between the lower and upper layers ofthe solvent system heptane -- CH₂ Cl₂ --CH₃ CN (50:15:35). The lowerlayer was concentrated to an oil (5.76 g), which was partitioned againbetween the upper and lower layers of the solvent systemEtOAc-heptane-MeOH-water (7:4:4:3). The lower layer, showing strongactivity, yielded a solid (800 mg), which was partitioned again betweenthe upper and lower layers of the solvent systemEtOAc-heptane-MeOH-water (7:4:4:3). The lower layer, showing strongactivity, yielded a solid (800 mg), which was then chromatographed togive four fractions over an RP silica gel gravity column withMeOH-aqueous NaCl (0.4 M) (7:1).

The first and most active fraction (333 mg) was separated by CCC intoten fractions with EtOAc-benzene-MeOH-cyclohexane-water (3:4:4:4:3) byusing the upper layer as a mobile phase. Fraction 7, containing Et 736as the major component, was separated by silica gel (treated with NH₃)column chromatography with CHCl₃ -MeOH (12:1). The first fraction (30.4mg) was purified by C-18 HPLC with CH₃ CN-MeOH-aqueous NaCl (0.25 M)(5:7:3) to give colorless needles (from CH₃ CN-H₂ O) of 3 (25 mg, 1.3×10⁻⁴ %): m.p. 140-150° C. dec., IR (CCl₄) 3530, 3480 (NH, OH) 2934,1768 (C═O), 1753 (C═O), 1196, 1153, 1089 cm⁻¹ ; IR (film) 3350, 3200(NH, OH), 2928, 1753 (C═O), 1440, 1250, 1200, 1088 cm⁻¹ ; [α], seeabove; NMR, see Table I; HRFABMS, see Table II.

Fraction 9 of the CCC separation was chromatographed on a silica gel(NH₃ treated) column with CHCl₃ --MeOH (8:1). The first fraction of thischromatogram was purified by the HPLC system described above to givelight-brown solid 4 (4 mg, 2.1 ×10⁻⁵ %); m.p. 160-164° C., IR (film)329l (NH, OH), 2930, 1753 (C═)), 1440, 1238, 1200, 1086 cm⁻¹ ; [α], seeabove; NMR, see Table I.

Fraction 10 (200 mg), most polar of the CCC separation, was furtherseparated into five fractions by CCC with CHCL₃ --MeOH--H₂ O (4:4:3),using the lower phase as the mobile phase. Fraction 5 (51 5 mg) of thisCCC run was separated on a silica gel (50 g) column with CHCl₃--MeOH--H₂ O (30:20:4) into 11 fractions. Of these, fraction 7 gavecrystalline tryptamine hydrochloride (7 mg); m.p. 230° C. dec. (lit,248° C., see, Merck Index, 1989 1540); TLC behavior and spectral dataidentical with those of an authentic sample (Aldrich).

The present invention has been described in detail, including thepreferred embodiments thereof. However, it will be appreciated thatthose skilled in the art, upon consideration of the present disclosure,may make modifications and/or improvements on this invention and stillbe within the scope and spirit of this invention as set forth in thefollowing claims.

What is claimed is:
 1. Ecteinascidin 736, essentially free of cellularmaterial of E. turbinata, and having the structural formula: ##STR3##wherein X ═OH and R ═CH₃.
 2. Ecteinascidin 722, essentially free ofcelluar material of E. turbinata, and having the structural formula:##STR4## wherein X ═OH and R ═H.
 3. O-Methyl-ecteinascidin 736,essentially free of cellular material of E. turbinata, and having thestructural formula: ##STR5## wherein X ═OCH₃ and R ═CH₃.